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Fig S1 Relative mRNA expression MCF7 pS2 * * * * * CCNG2 * * * * * * RPRM * * * * KLF6 * * * * * * CLDN4 * * * CERK * * * EFEMP1 * * * ENC1 * * BIK * *

Published byGeorgiana Lewis Modified over 8 years ago

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Presentation on theme: "Fig S1 Relative mRNA expression MCF7 pS2 * * * * * CCNG2 * * * * * * RPRM * * * * KLF6 * * * * * * CLDN4 * * * CERK * * * EFEMP1 * * * ENC1 * * BIK * *"— Presentation transcript:

1 Fig S1 Relative mRNA expression MCF7 pS2 * * * * * CCNG2 * * * * * * RPRM * * * * KLF6 * * * * * * CLDN4 * * * CERK * * * EFEMP1 * * * ENC1 * * BIK * * * * * * HBP1 * * * * * CXCR4 * * * * RAP1GA1 * * * * CDH3 * * BMP7 * * MXD4 * * * * MUC1 * * * * NDRG1 * * * RBL2 * BAKp21PLK2 * DUSP4 * * * 4 hr8 hr24 hr4 hr8 hr24 hr4 hr8 hr24 hr4 hr8 hr24 hr Fig S1: Identification of estrogen-repressed genes in MCF7 cells. q-RT-PCR was used to confirm the estrogen-mediated repression of candidate genes identified from microarray data. The relative mRNA expression is depicted as ligand-mediated fold change compared to vehicle control. MCF7 cells were treated for 4, 8, or 24 hours with either vehicle, estrogen (E2), or a combination of E2 and ICI (E2+ICI). The data are an average of three replicates + SEM. A t-test analysis was performed in which the E2 treatment group was compared to the vehicle group and the E2+ICI group was compared to the E2 group (* p < 0.05).

2 Fig S2 Fig S2: Estrogen-mediated repression of selected genes in breast cancer cells. q-RT-PCR was used to confirm the estrogen-mediated repression of candidate genes identified from microarray data. The relative mRNA expression is depicted as ligand-mediated fold change compared to vehicle control. (A) ZR-75-1 and (B) T47D cells were treated for 4, 8, or 24 hours with either vehicle, estrogen (E2), or a combination of E2 and ICI. The data are an average of three replicates + SEM. A t-test analysis was performed in which the E2 treatment group was compared to the vehicle group and the E2+ICI group was compared to the E2 group (* p < 0.05). Relative mRNA expression ZR-75-1 CERK 4 hr8 hr24 hr RPRM * * * KLF6 * * CLDN4 * * * EFEMP1 * 4 hr8 hr24 hr ENC1 4 hr8 hr24 hr A B Relative mRNA expression T47D RPRMKLF6 CERKEFEMP1 ENC1 CLDN4 * * * * * * * * * 4 hr8 hr24 hr4 hr8 hr24 hr4 hr8 hr24 hr

3 Fig S3 Fig S3: Identification of E2-repressed genes that are primary ERα targets. (A) MCF7 cells were treated with either vehicle, estrogen (E2), or a combination of E2 and cycloheximide (CHX). q-RT-PCR was used to calculate relative mRNA expression as ligand- mediated fold change compared to vehicle control. This fold change is reflected in the color intensity as shown in the legend. A 4h8h E2 CHX ++++ -++- E2 Fold change <0.3 0.3-0.5 0.5-0.7 0.7-0.9 1.3-1.5 1.5-1.9 >2 1.1-1.3 0.9-1.1 CLDN4 EFEMP1 CERK KLF6 ENC1 BIK HBP1 CXCR4 RAP1GA1 CDH3 BMP7 MXD4 NDRG1 RBL2 FLOT1 RND3 TENS1 IER3

4 Fig S4 Fig S4: Knockdown of HDAC1-10 and its effect on RPRM expression. MCF7 cells were transfected with non-specific siRNA (siNS) or siRNA against HDAC1-10 (siHDAC1-10) followed by either a vehicle or an E2 treatment for 12 hrs. The knockdown of each HDAC is shown in (A) and its effect on RPRM expression is shown in (B). q-RT-PCR was used to calculate relative mRNA expression as fold change compared to vehicle control. The data are an average of three replicates + SEM. A HDAC1HDAC2HDAC3HDAC4HDAC5 HDAC6HDAC7HDAC8HDAC9HDAC10 Relative mRNA expression siNSsiHDAC1siNSsiHDAC2siNSsiHDAC3siNSsiHDAC4siNSsiHDAC5 siNSsiHDAC6siNSsiHDAC7siNSsiHDAC8siNSsiHDAC9siNSsiHDAC10 B RPRM Relative mRNA expression siNSsiHDAC1siNSsiHDAC2siNSsiHDAC3siNSsiHDAC4siNSsiHDAC5 siNSsiHDAC6siNSsiHDAC7siNSsiHDAC8siNSsiHDAC9siNSsiHDAC10

5 Fig S5 Fig S5: Localization and expression of the HDAC7 deletion and mutant constructs. (A) 293 cells were transfected with Flag-HDAC, Flag-HDAC7 (1-487), Flag-HDAC7 (438-912), and Flag-HDAC7 (H670A) and immunostained for Flag. (B) 293 cells were transfected with Flag-HDAC, Flag-HDAC7 (1-487), Flag-HDAC7 (438-912), and Flag-HDAC7 (H670A) and blot for Flag and β-actin. B HDAC7: +---- --+- HDAC7 (1-487): - HDAC7 (438-912): ---- + HDAC7 (H670A): ---+ - β-actin Flag 100 75 45 50 150 MW (kDa) HDAC7HDAC7(1-487)HDAC7 (438-912)HDAC7 (H670A) A

6 * A SYBR Green assay was used for genes lacking a probe. *

7

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